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Characterization of an extracellular serine protease of Leishmania (Leishmania) amazonensis

Published online by Cambridge University Press:  28 February 2005

R. E. da SILVA-LOPEZ ,
M. G. PINTO COELHO  and
S. G. De SIMONE
R. E. da SILVA-LOPEZ
Affiliation:
Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil
M. G. PINTO COELHO
Affiliation:
Departamento de Bioquímica, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brasil
S. G. De SIMONE
Affiliation:
Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Rio de Janeiro, RJ, Brasil Departamento de Bioquímica e Biologia Molecular, Instituto de Biologia, Universidade Federal Fluminense, Rio de Janeiro, Niterói, Brasil
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Abstract

A serine protease was purified 942-fold from culture supernatant of L. amazonensis promastigotes using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin-agarose and continuous elution electrophoresis by Prep Cell, yielding a total recovery of 61%. The molecular mass of the active enzyme estimated by SDS-PAGE under conditions of reduction was 56 kDa and 115 kDa under conditions of non-reduction, suggesting that the protease is a dimeric protein. Additionally, it was found to be a non-glycosylated enzyme, with a pI of 5·0. The optimal pH and temperature of the enzyme were 7·5 and 28 °C respectively, using α-N-ρ-tosyl-L-arginine-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 61% of the enzyme activity was preserved after 1 h of pre-treatment at 42 °C. Haemoglobin, bovine serum albumin (BSA), ovalbumin, fibrinogen, collagen, gelatin and peptide substrates containing arginine in an ester bond and amide substrates containing hydrophobic residues at the P1 site were hydrolysed by this extracellular protease. The insulin β-chain was also hydrolysed by the enzyme and many peptidic bonds were susceptible to the protease action, and 4 of them (L11-V12, E13-A14, L15-Y16 and Y16-L17) were identified. Inhibition studies suggested that the enzyme belongs to the serine protease class inhibited by calcium and manganese and activated by zinc. These findings show that this enzyme of L. amazonensis is a novel serine protease, which differs from all known flagellate proteases characterized.

Keywords

Leishmania amazonensisextracellular serine proteasepurificationcharacterization
Type
Research Article
Information
Parasitology , Volume 131 , Issue 1 , July 2005 , pp. 85 - 96
Copyright
© 2005 Cambridge University Press

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